1. Technical Field
The present invention relates to optical testing and measuring using dispersed light spectroscopy including Raman scattering. The present invention particularly relates to optical testing and measuring based on linear and non-linear optical emissions from an active separation column delivering enhanced molecularly related information pertaining to the contents of the column.
2. General Background
Molecular separation columns have been used for many years to separate and analyze sub-molar, milli-molar, and even micro-molar concentrations of specific organic molecules. Examples of such molecular separation columns include chromatography, electrophoresis, flow cytometry, HPLC, and density gradient separation. As the concentration of a specific organic molecule decreases, the sensing of a reliable indicator for that target molecule becomes increasingly difficult. Much work has been done toward enhancing detection capability so that even smaller molar concentrations can be used as acceptable samples. The ultimate ideal, of course, is to be able to reliably detect the presence of a single target molecule in a given sample. A practical goal is to gather reliable information concerning molecules that are present in nano-molar, pico-molar, and even femto-molar concentrations in a given sample.
A number of techniques are available for gathering information concerning the nature of the molecules in a given sample by virtue of indicative spectral characteristics including UV spectroscopy, IR spectroscopy, Brillouin scattering, Raman scattering, fluorescence spectroscopy, multi-photon fluorescence spectroscopy and mass spectrometry. Surface Enhanced Raman Scattering (SERS), including hyper-Raman scattering, is of particular interest as it tends to give large enhancements of characteristic Raman spectra when the specimen is in the near vicinity of certain materials that can be generally characterized as coinage metals, e.g., gold, silver, copper, nickel, and aluminum in the form of particles or films.
The general techniques of SERS is well established and is discussed to varying degrees in a variety of references ranging from text books to additional patents such as U.S. Pat. No. 5,693,152 to Carron, U.S. Pat. No. 5,255,067 to Carrabba, U.S. Pat. Nos. 5,266,498, 5,376,556, and 5,567,628 to Tarcha, and U.S. Pat. No. 6,608,716 to Armstrong, et al. To the extent necessary, each of these references is hereby incorporated by reference to provide additional understanding as they relate to the Raman and SERS techniques generally.
Optical microcavities are generally dielectric resonant structures that have at least one dimension that is at least on the order of about 10−6 meters up to about 10−2 meters. The specific geometry of the microcavity and the boundary conditions on any interface of the dielectric to an adjacent medium impose selective normal modes on the optical microcavity, sometimes referred to as morphology-dependent resonances (MDRs). Such microcavities have been employed at least experimentally to construct light emitting devices. Further, resonant microcavities can emit light in a highly directional manner as a result of their inherent geometry. These resonances, which may have very high quality factors, Q on the order of 105 to 1010, result from confinement of the radiation within the microcavity by total internal reflection. Light emitted within or scattered in the microcavity may couple to high-Q MDRs lying within its spectral bandwidth, leading to enhancement of both spontaneous and stimulated optical emissions.
Resonant microcavities are known to cause large enhancements of optical emissions. For example, enhanced fluorescence emission from a dye-doped cylindrical or spherical microcavity occurs when either the laser pump or the fluorescence, or both, couple to microcavity MDRs. J. F. Owen, Phys. Rev. Lett. 47, 1075 (1981). Moreover, the increased feedback produced by MDRs is sufficient to obtain laser emission from a dye-doped microdroplet under both a continuous wave (CW) and pulsed laser excitation. H. M. Tzeng, et al., Opt. Lett. 9, 499 (1984); A. Biswas, et al., Opt. Lett. 14, 214 (1988). The existence of high-Q microcavity modes is also responsible for numerous stimulated nonlinear effects including stimulated Raman and Rayleigh-wing scattering and four-wave parametric oscillation under moderate intensity CW excitation. M. B. Lin and A. J. Campillo, Phys. Rev. Lett. 73, 2440 (1994).
Direct on-column SERS detection of 10−6 M Riboflavin and 10−8 M Rhodamine 6G has been demonstrated in capillary electrophoresis incorporating buffers containing silver colloidal solutions. The capillaries were 100×10−6 m inside diameter fused-silica capillaries with an outside diameter of 365×10−6 m. At the reported molar concentrations for the test compounds, acquisition of satisfactory spectral data could be obtained using a 17 mW laser operating at 515 nm wavelength in about one second. W. F. Nirode, et al., Anal. Chem. 72, 1866 (2000). For lower molar concentrations, commensurately longer data acquisition times are expected. For sub-nanomolar concentrations, the time expected for sufficient data acquisition increases so that so called on-the-fly acquisition is unlikely without other changes to the system.
What is still needed is an instrument for multiplexed protein analysis with enhanced sensitivity and lower recurring chemical costs compared to current proteomic detection and labeling technologies, which is operable on nano-molar and pico-molar, and in some cases even femto-molar concentrations of a given protein in question.